猪伪狂犬病病毒TK／gE双基因缺失株PRV／TK^-／gE^-的构建及其生物学特性  

Construction and biological characterization of TK/gE-null recombinant pseudorabies virus

作　　者：邵定勇 黄红亮 唐兆新[1,2] 任常宝 谢小雨 陈瑞爱[1,2] 

机构地区：[1]肇庆大华农生物药品有限公司广东省兽用生物制品生物技术研究与应用企业重点实验室,广东大旺526238 [2]华南农业大学兽医学院,广东广州510642

出　　处：《中国兽医科学》2013年第11期1127-1132,共6页Chinese Veterinary Science

基　　金：肇庆市科技计划项目(2012C003)

摘　　要：为了构建伪狂犬病病毒新兴株(PRV-XX)TK/gE双基因缺失的重组病毒,利用PCR从PRV-XX株基因组分别扩增gE基因两侧序列LgE和RgE,将LgE和RgE克隆到pUC19载体,构建转移质粒pUC19-gE/HEK;进一步将增强型绿色荧光蛋白基因(EGFP)表达盒克隆到质粒中,构建转移质粒pUC19-gE/EGFP;先用构建好的pUC19-TK/EGFP质粒与PRV-XX基因组共转染BHK21细胞,噬斑筛选纯化获得重组病毒PRV/TK-/EGFP;再用pUC19-TK/HEK与PRV/TK-/EGFP病毒基因组共转染BHK21细胞,噬斑筛选纯化获得重组病毒PRV/TK-。在此基础上,用同样方法获得重组病毒PRV/TK-/gE-/EGFP和PRV/TK-/gE-。重组病毒PRV/TK-/gE-的生物学特性研究结果表明:TK/gE缺失稳定,30代次内未见缺失位点变化;TK/gE缺失后,重组病毒PRV/TK-/gE-毒力是亲本毒株PRV-XX株的1/48,但不影响其在ST细胞上的增殖;PRV/TK-/gE-对猪安全有效,其最小免疫剂量为105.0 TCID50,免疫水平与某进口伪狂犬病疫苗相当。The purpose of this study was to construct TK/gE-null recombinant pseudorabies virus （PRV） of PRV-XX strain. Firstly, transfer vector pUC19-gE/HEK was constructed using both ends of PRV gE gene, which were successfully amplified by PCR from PRV-XX genome, and then the enhanced green fluorescent protein（EGFP） gene expression box was cloned into pUC19-gE/HEK,resulting in a uni- versal transfer vector pUC19-gE/EGFP. After cotransfecting pUC19-TK/EGFP and genome of PRV-XX into BHK21 cells,recombinant virus PRV/TK^-/EGFP was obtained following plaque screening, pUC19- TK/HEK and genome of PRV/TK^-/EGFP were cotransfected into BHK21 cells to obtain recombinant virus PRV/TK^-. Recombinant viruses PRV/TK^-/gE^-/EGFP and PRV/TK^- /gE^- were also obtained as the above mentioned method. The results of PRV/TK^-/gE^- biological characteristics research showed as follows：the deletions of TK and gE genes were genetic stable. Compared with the parent virus, there was no mutation between PRV/TK^- /gE^- and PRV-XX in 30 passages. Both of the PRV/TK^- /gE^- and PRV- XX had the same ability to affected the ST cells,but the virulence of PRV/TK^- /gE^- decreased to 1/48 of PRV-XX. PRV/TK^-/gE^- was safe and effective to swine. It showed the immunity level as the same as that induced by one imported vaccine,and the minimum dose of PRV/TK^- /gE^- was 10^5.0 TCID50.

关 键 词：伪狂犬病病毒XX株 TK gE双基因缺失 生物学特性 

分 类 号：S852.659.5[农业科学—基础兽医学]